Tetrahydrofuran (THF) is a toxic and carcinogenic compound that is commonly released from pharmaceutical, chemical and related industry wastewater. Currently, the effects of THF contamination on wastewater are unknown and a better understanding of THF toxicity toward biological processes in wastewater treatment is critical. In this study, we firstly investigated the toxic effects of THF on enzymatic activity and the microbial diversity in activated sludge from a sequencing batch reactor during long-term exposure to 10 mM THF. The activity of five enzymes (catalase, dehydrogenase, urease, phosphatase and protease) was remarkably decreased in the presence of 10 mM THF during a period of 85 days. Of these five affected enzymes, dehydrogenase activity was close to detection level limits and was nearly completely inhibited. Analysis of the microbial community demonstrated that THF, at a concentration of 10 mM, altered the distribution of microbes within the community and significantly decreased microbial diversity during long-term contamination, according to denaturing gradient gel electrophoresis (DGGE) analysis. The fraction of Actinobacteria increased in the community, while the fraction of Proteobacteria significantly decreased after THF exposure.